Cardiovascular disease (CVD) is the leading cause of death in most industrialized nations of the world. In the United States alone, 61 million people comprising approximately 25% of the total population have been reported to suffer from some type of CVD. Reducing the levels of blood cholesterol, in particular the low-density lipoprotein cholesterol, is known to be one of the effective strategies for the prevention of CVD [1].
A class of drugs, called statins, has been found to be highly effective in the reduction of blood cholesterol levels. Statins work by suppressing the activity of HMG-CoA reductase, the rate limiting enzyme in the synthesis of cholesterol in cells. The resultant decrease in intracellular concentrations results in a compensatory up-regulation of surface low-density lipoprotein (LDL) receptors, which in turn mediates clearance of LDL-cholesterol from plasma. Clinically, HMG-CoA reductase inhibitors are effective in lowering blood LDL-cholesterol levels by 20-40% and have also been found to be effective in reducing the levels of triglyceride and inducing modest increases in the levels of HDL-cholesterol in blood [2,3]. Despite their success in the management of blood lipid concentrations, statin therapy however cannot be prescribed to all patients with hyperlipidemia. In particular, these drugs are contraindicated in patients with cholestasis or impaired hepatic functions. In some patients, statins have also been reported to cause elevations in various hepatocellular enzymes such as creatine kinase. Statins have also been found to have potential adverse effects on muscle, kidney and the nervous system of some patients. There have also been reports of myopathy during the treatment with statins. Although myopathy occurs in only 0.1 to 0.2% of patients receiving statins, its risk increases with the increase in dose. In addition, if left untreated, myopathy could lead to a more serious and life-threatening condition called rhabdomylosis. Consequently, following the development of myopathy immediate cessation of statin therapy is required [4,5].
Treatment of hypercholesterolemic patients with berberine (BBR) at a dose of 1 g/day for 3 months was found to reduce serum cholesterol concentrations by 29%, triglycerides by 35% and LDL-cholesterol by 25% [6]. Experimental studies also support the cholesterol-lowering efficacy of BBR. Oral administration of BBR to hyperlipidemic hamsters at a dose of 100 mg/day for 2 weeks was found to reduce serum total- and LDL-cholesterol levels by up to 30% [6-8]. A number of in vitro studies utilizing human hepatoma cell lines such as Hep G2 cells have also been carried out to determine the underlying mechanisms of cholesterol-lowering effects of BBR. Results from these studies indicate that BBR increases the expression of LDL receptors in the cells through a post-transcriptional mechanism that increases the stabilization of mRNA for these receptors. BBR was also found to inhibit the synthesis of cholesterol and triglyceride in the cells through activation of an AMP-activated protein kinase [6-8]. In a more recent study, BBR was found to inhibit differentiation of 3T3-L1 adipocytes and to inhibit lipogenesis in the cells through PPAR pathways [9].
Although evidence from a number of studies indicates that BBR has cholesterol-lowering properties, a dose of approximately 1 g/day appears to be required to achieve clinical benefits. Safety of chronic administration of this dosing remains a concern. In an experimental study, intra-peritoneal administration of BBR at doses exceeding 15 mg/Kg for 10 days to mice was found to be lethal to the animals [10]. Higher doses of BBR were also found to be toxic to cells in culture. For example, treatment of L929 cells with BBR in doses greater than 40 μg/ml was found to induce cytotoxicity. More recently, incubation of human liver microsomes with BBR at concentrations of 20 μM was found to decrease the activities of various CYP 450 enzymes including CYP2D6, CYP3A4 and CYP2E1 [11].
An herbal tea, Ku Ding Cha (also referred to as Kudingcha, Kuding tea, bitter tea, ku cha), is also known to have cardiovascular benefits. It is widely consumed in China as a normal tea or functional drink. The original plant of Ku Ding Cha consists of about 10 most commonly known species in the genus Ligustrum, Oleaceae, and Ilex, Aquifoliaceae including Ligustrum pedunclare, Ligustrum purpurascens, Ligustrum japonicum, Ligustrum robustrum, Ilex cornuta, Ilex kudincha C. J. Tseng (Aquifoliaceae), Ilex latifolia, Cratoxylum prunifolium, Ehretia thyrsiflora, Photinia serruiata [12] and Ilex paraguariensis. The extract from these ground plants in hot water is consumed as a tea. In animal studies extracts of some of these species have been found to promote circulation of blood, lower blood pressure, have anti-oxidative effects and reduce levels of lipids in plasma [13-15]. For example, in an experimental study daily gavaging of water extracts of Ilex paraguariensis to hypercholesterolemic rats at a dose of 500 mg/day for 2 weeks was found to significantly reduce the plasma concentrations of cholesterol and triglycerides [16]. In another study, daily administration of extracts of Ligustrum japonicum for 1-3 months reduced the levels of blood total cholesterol in hypercholesterolemic rabbits [17]. The clinical benefits of the tea in the management of human blood cholesterol concentrations however have not been determined.
The biologically active components of Ku Ding Cha have yet to be determined. Aqueous and alcoholic extracts of leaves of various Ilex species contain numerous terpenoids, flavonoids [18-23], triterpenes, phenylethanoid glycosides, caffeoyl quinic acid and derivatives thereof. Some of these compounds are implicated in the cholesterol-lowering effects of Ku Ding Cha; recent studies have found that a number of mono- and tri-terpenes isolated from the leaves and twigs of two species of Ku Ding Cha including Ilex kudingcha and Ilex macropoda were found to inhibit the activity of acyl-CoA: cholesterol acyltransferase (ACAT) in vitro [24-27]. ACAT activity is important for regulation of cholesterol absorption from the gut and esterification of cholesterol with fatty acids in mammals. Inhibition of ACAT in hepatocytes has been shown to decrease the secretion of apolipoprotein-B containing lipoproteins, such as very low density lipoprotein (VLDL) particles from the liver [28].
Many of the triterpenoid saponins have been isolated from Ilex kudingcha: ilekudinosides A-S, ilexoside XL VIII, cynarasaponin C, latifolosides A, C, G and H, kudinoside G. Some of them exhibited significant ACAT inhibitory activity [26].
Some monoterpenoid and phenylethanoid glycosides have also been isolated from Ku Ding Cha (Ligustrum pendunculare) leaves: lipedosides A-I and A-II as phenylthanoid glycosides, and lipedosides B-I, B-II, B-III, B-IV, B-V, and B-VI as monoterpenoid glycosides. Lipedoside B-III has been identified as inhibitor of ACAT with IC50 of 269 μM [23]
Ku Ding Cha is also known to contain ursolic acid, betulin, lupeol, and chlorogenic acid.
Ursolic acid (3β-Hydroxy-12-ursen-28-ic acid), has been identified as an inhibitor of several enzymes, including adenosine deaminase, arachidonate lipoxygenase, aromatase, cyclooxygenase, DNA ligase I, elastase, protein kinases A and C, and RNA-directed DNA polymerase [29]. Recently, ursolic acid has also been identified as an inhibitor of nucleoside triphosphate hydrolase (NTPase, IC50 0.6 μM) and acyl-CoA: cholesterol acyltransferase (ACAT, IC50, 58.8 μM) [30-31]. Ursolic acid is also a strong antitumorigenic and chemopreventive agent [32].
Betulin (lup-20[29]-ene-3β,28-diol) and betulinic acid (3-hydroxy-20[29]-lupen-28-oic acid) are anti-inflammatory and cytotoxic against a variety of tumor cell lines [33]. U.S. Pat. No. 5,679,828 has found that betulinic acid and its derivatives have strong anti-HIV infection activity. Betulinic acid has also been identified as an inhibitor of acyl CoA: diacylglycerol acyltransferase (DGAT, IC50, 9.6 μM) [34]. Furthermore, betulin and betulinic acid are good ACAT inhibitors with IC50 of 83 and 16.2 μM, respectively [24].
Lupeol (3β-Hydroxy-20(29)-lupene, or 20(29)-Lupen-3β-ol) has been found to have anti-cancer activity via down-regulation of NF-kB and has been identified as an inhibitor of acyl-CoA: cholesterol acyltransferase (ACAT IC50, 48 μM) [24,35].
Chlorogenic acid, a chemical component of Ku Ding Cha, has been identified as squalene synthase inhibitor, with an IC50 of 0.1 μM [36].
Hawthorn, a plant cultivated widely in Europe and China (called shanzha in China), has been found to have various health-promoting properties. In Europe, the species of hawthorn which are commonly accepted in herbal medicine include Crataegus oxyacantha, Crataegus pentagyna Waldst, Crataegus nigra Waldst, Crataegus azarolus L. and Crataegus monogyna. In China, two other varieties of hawthorn including Crataegus pinnatifida and Crataegus pinnatifida Bge var. major NE Br., have been used in traditional Chinese medicine. Studies indicate that both European as well as Chinese varieties have various health benefits including anti-oxidant, anti-inflammatory and hypolipidemic effects. These varieties have also been found to have protective effects on the brain and vascular endothelium and endothelium-dependent relaxation. In addition, some clinical studies also indicate that hawthorn has blood pressure lowering properties and is effective in the treatment of mild forms of arrhythmia. Flavonoids, in particular proanthocyanidines are believed to be the active components of hawthorn. Studies utilizing various flavonoids indicate that these compounds possess cholesterol-lowering properties and more specifically they have been found to increase the hepatic uptake of cholesterol [37].